THE   GROWTH   OF  CITRUS   SEEDLINGS   AS 

INFLUENCED  BY  ENVIRONMENTAL 

FACTORS1 


BY 

RAYMOND  E.  GIRTON 


CONTENTS 

Experimental  technique 83 

Environmental  factors 85 

Temperature 85 

Hydrogen-ion  concentrations 90 

Oxygen 95 

Mixtures  of  gases 101 

General  discussion 113 

Summary 114 

Literature  cited.. 116 

Environment  plays  a  large  part  in  the  life  of  plants  as  well  as  of 
human  beings.  Of  the  many  factors  of  environment,  those  selected 
for  the  present  investigation  were  temperature,  hydrogen-ion  concen- 
trations, oxygen,  and  mixtures  of  gases. 


EXPERIMENTAL  TECHNIQUE 

The  experiments  were  conducted  under  continuous  artificial  illumi- 
nation and  an  effort  was  made  to  maintain  uniform  temperatures  and 
humidity.  The  first  series  of  experiments  was  conducted  in  a  saturated 
atmosphere,  but  all  subsequent  experiments  were  carried  out  under  a 
relative  humidity  of  approximately  50  to  60  per  cent.  In  addition, 
control  cultures  were  maintained  whenever  possible  in  order  to  provide 
an  adequate  basis  for  comparison. 

The  periods  over  which  these  experiments  were  continued  were  not 
long,  since  two  weeks  usually  sufficed  to  bring  out  distinct  differences 
between  the  root  growth  of  the  treated  plants  and  that  of  the  controls. 


1  Paper  No.  146,  University  of  California,  Graduate  School  of  Tropical  Agri- 
culture and  Citrus  Experiment  Station,  Riverside,  California. 


84 


University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 


Ordinarily  this  period  also  allowed  time,  under  favorable  growth  con- 
ditions, for  the  production  of  a  second  pair  of  leaves.  All  roots  were 
coated  lightly  with  a  suspension  of  carbon  black  when  the  experiments 
were  set  up.  This  made  it  possible  to  distinguish  all  subsequent 
growth  by  its  natural  whitish  color  as  contrasted  with  the  darker 
coated  regions. 

When  solution  cultures  were  required,  a  form  of  Hoagland's 
nutrient  solution  (pH  5.2)  was  used.  This  solution  was  modified  by 
the  use  of  a  half-and-half  mixture  of  mono-  and  dibasic  potassium 
phosphates  instead  of  the  monobasic  form  alone  as  ordinarily 
employed.  Thus  a  less  acid  reaction  was  obtained  (pH  6.0)  which  was 
more  favorable  to  the  growth  of  citrus  roots.  The  solutions  were  not 
changed  during  the  experiments,  but  iron  was  added  two  or  three 
times  each  week  in  the  form  of  a  dilute  solution  of  ferric  tartrate. 

The  production  of  root  hairs  was  measured  by  means  of  an  index 
expressing  their  relative  abundance.  This  index,  called  the  'root- 
hair  index, '  indicated  the  average  number  of  root  hairs  per  centimeter 
along  one  side  of  a  rootlet.  This  index  was  obtained  in  the  following 
manner.  Estimates  were  made  of  the  relative  abundance  of  root 
hairs  by  classifying  each  plant  examined  on  the  basis  of  the  following 
classes :  I  =  very  few  root  hairs,  II  =  few,  III  s=  moderate  number, 
IV  =  abundant,  and  V  =  very  abundant.  It  was  found  by  actual 
count  that  the  average  root-hair  indexes  for  these  classes  had  the 
following  approximate  values  (see  table  1)  :  I  =  5,  II  =  15,  III  =  30, 
IV  =  60,  and  V  =  120. 

TABLE   1 
Approximate  r.  h.  i.   (root-hair  index)   Kelationships 


Values  of  r.h.i.  determined  by  actual  count 

Approximate 

Class 

Number  of  plants 
examined 

Index 

r.h.i. 
(assigned  values) 

I 

20 
43 
40 
16 
2 

4.5±0  4 

14.5  ±0.5 

33.9  ±  1  4 

59  8±4  4 

119 

5 

11 

15 

III 

30 

IV 

60 

V 

120 

The  approximate  root-hair  index  for  a  given  population  of  plants 
could  therefore  be  determined  by :  first,  a  microscopic  examination  in 
which  each  plant  was  classified  according  to  the  estimated  abundance 
of  root  hairs;  second,  the  number  of  plants  within  each  class  was 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment  85 

multiplied  by  the  assigned  index  value  for  the  class ;  and  third,  the 
resulting  products  were  added  and  the  sum  divided  by  the  total  num- 
ber of  plants.  This  gave  a  quantitative  index  for  the  relative  abun- 
dance of  root  hairs  which  was  based  upon  estimates  of  the  individual 
plants.  The  following  formula  expresses  in  a  condensed  form  the 
relationships  just  discussed. 

_  2  (ai .  ni  -+-  an  .  nn -+-  av  .  nv) 

Ia~  N 

Ia  =  approximate  root-hair  index. 

ai,  an,  etc.  =  average  root-hair  indexes  for  the  respective  groups 

I,  II,  etc. 
iii,  nn,  etc.  =  number  of  plants  falling  within  the  designated  group. 

N  =  total  number  of  plants  producing  root  elongation  under  the 
experimental  conditions. 

A  simple  example  may  serve  to  illustrate  the  method.  Suppose 
that  in  a  population  of  30  plants,  it  was  determined  by  a  microscopic 
examination  of  the  roots  that  the  plants  should  be  classified  as  follows : 
3  in  class  I,  7  in  class  II,  15  in  class  III,  and  5  in  class  IV.  Then  the 
approximate  values  for  the  different  classes  would  be :  3  (the  number 
of  individuals  in  the  class)  x  5  (the  approximate  r.h.i.  for  the  class) 
=  15  for  class  I,  7  x  15  =  105  for  class  II,  15  x  30  =  450  for  class 
III,  and  5  x  60  =  300  for  class  IV.  The  sum  of  these  products  (870) 
divided  by  the  total  number  of  plants  (30)  gives  the  approximate 
root-hair  index  for  the  entire  population  (29).  Substitution  in  the 
suggested  formula  would  give  the  same  result. 

T         S  (3.5 +  7.15 +  15.30 +  5.60) 
Ia~~  ~30~  "  =  29 


ENVIRONMENTAL  FACTORS 
Temperature 
Seedlings  of  the  grapefruit  {Citrus  maxima  (Burm.)  Merrill),  the 
sour  orange  (Citrus  aurantium  L.),  and  the  sweet  orange  (Citrus 
sinensis  Osbeck)  were  grown  at  constant  temperatures  in  a  differential 
thermostat  placed  at  the  writer's  disposal  by  the  Division  of  Plant 
Pathology  of  the  Citrus  Experiment  Station.  This  apparatus  is 
similar  to  the  one  described  by  Livingston  and  Pawcett  (1920).  It 
contains  seven  cylindrical  compartments,  40  centimeters  in  diameter 
and  45  centimeters  in  depth,  with  maintained  temperatures  graded 
from  about  12°  to  35°  C,  depending  upon  the  adjustment  and  the 


86 


University  of  California  Publications  in  Agricultural  Sciences       [Vol.  5 


manner  of  illumination.  Each  compartment  was  covered  with  a  lid 
made  of  two  thicknesses  of  window  glass  with  a  3-centimeter  air  space 
intervening.  An  electric  light  suspended  above  each  compartment 
furnished  continuous  illumination.  In  the  first  trial,  with  grapefruit 
plants,  75-watt  lamps  were  employed,  but  these  were  too  weak  for  the 
best  photosynthetic  activity  and  were  replaced  by  250-watt  lamps  in 
the  subsequent  experiments. 


GEAPEFRUIT  SEEDLINGS 

Four-  to  five-weeks-old  seedlings  were  selected  for  comparative  uni- 
formity of  top  and  root  development.  They  were  supported  in 
paraffined  cork  stoppers  and  placed  in  one-quart  jars,  each  culture 
jar  containing  six  seedlings.  Five  jars,  containing  a  total  of  30  plants, 
were  then  placed  in  the  apparatus  and  maintained  at  the  following 
temperatures:  11°,  UV20,  19°,  22V20,  26°,  30°,  and  34°  C,  respectively. 
Continuous  illumination  was  supplied  by  75-watt  'daylight'  electric 
lamps  suspended  about  one  meter  above  the  plants,  one  lamp  to  each 
compartment.  Tbe  plants  were  removed  at  the  end  of  the  third  week 
and  examined  for  increase  in  root  length  and  production  of  root  hail's 
(table  2).  The  examination  showed  that  the  root  growth,  which  was 
confined  to  elongation  of  the  tap  roots,  and  the  root-hair  production 
were  greatest  at  26°  C  and  consistently  decreased  with  higher  or  lower 
temperatures.  The  course  of  the  growth  is  shown  by  the  graphs  in 
figures  1  and  2. 


TABLE   2 

Root  Growth  of  Grapefruit   Seedlings   at   Different   Temperatures  for  a 

Three-weeks'  Period 


Usual 

Temperature 

Number  of 

Entire  root 

Root 

Root-hair 

temperature 

fluctuations* 

plants 

elongation 

per  plant 

index 

°C 

0  C 

cm. 

cm. 

11 

10    -lti' , 

30 

2.9 

0  10 

7 

14H 

13 ',-19 

30 

10.8 

0.36 

8 

19 

18    -22 

30 

10  8 

0  36 

24 

22H 

21',  25 ' j 

30 

19  2 

0  64 

47 

26 

25    -28' 0 

30 

30  2 

0  97 

78 

30 

2sy2-3\y2 

30 

23  7 

0  79 

58 

34 

32H-35H 

27 

9  2 

0  34 

8 

*  Temperature  fluctuations  recorded  in  this  column  were  due  in  part  to  the  stoppage  of  the  cooling 
apparatus  for  a  short  time. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment 


87 


/S-  10  25  17F 
Degrees  Cer>tr/'yrac/e.- 


fO 


Fig.  1.     Graphs  showing  average  root  growth  of  citrus  seedlings  grown 
at  various  maintained  temperatures. 


SOUK-ORANGE  SEEDLINGS 
It  may  be  seen  from  table  3  and  from  figures  1  and  2,  that  the 
total  root  elongation  of  the  sour-orange  plants  followed  the  trend 
previously  noted  for  the  grapefruit  seedlings,  i.e.,  increased  growth 
with  increased  temperature  up  to  a  point  of  maximum  growth  and  a 
rapid  decrease  in  growth  with  a  further  increase  in  temperature.  The 
elongations  of  the  tap  and  lateral  roots  were  roughly  parallel  over  the 
entire  temperature  range.  On  the  other  hand,  the  number  of  lateral 
roots  produced  showed  no  relation  to  the  temperature.  The  consid- 
erably greater  root  elongation  obtained  with  sour  orange  as  compared 
with  that  obtained  with  grapefruit  was  due  largely  to  the  increased 
illumination  in  the  sour-orange  experiment. 


88 


University  of  California  Publications  wj  Agricultural  Sciences      [Vol.  5 


The  production  of  root  hairs  exhibited  a  temperature  relation 
similar  to  that  of  root  elongation.  The  optimum  temperature,  however, 
was  somewhat  higher. 

TABLE  3 

Growth  of  Sour-orange  Seedlings  at  Different  Temperatures  for  a 

Two-weeks'  Period 


Temperature 

Num- 
ber 
of 

plants 

Stem 

Leaves 

Tap 
root 
elonga- 
tion 

Lateral  roots 

Total 
root 
elonga- 
tion 

Ap- 
proxi- 

Usual 

Fluctu- 
ations* 

Diam- 
eter 

Height 

Number 

Fresh 
weight 

Number 

Elonga- 
tion 

mate 
root- 
hair 

index 

°C 

•c 

cm. 

cm. 

gm. 

cm. 

cm. 

cm. 

13 

12H-16H 

30 

18±  002 

7.3±0  11 

2  1±0  01 

12±  004 

0  3±0.03 

2  6±0  36 

O.5±0.07 

0  8±0.10 

1±0.2 

18 

17^-20^ 

30 

18±  003 

7.8±0.12 

2  lzhO.01 

13±  003 

1.9±0  14 

11  1±0  67 

12  4±1  12 

14  3±1  17 

6±0  4 

23^ 

23    -25 

30 

.  18±  002 

7  9±0  13 

3  OdzO.lO 

13±  005 

3  8±0  22 

7  0±0  53 

13  Oil  28 

16.8±1  32 

13±1  4 

27 

2V/2-2%V2 

30 

17±002 

7  9±0  11 

3  5±0  08 

.  17±  008 

6  3±0  33 

7  1±0  63 

14.2±1.39 

20  5±1  43 

21±2  3 

31 

30>*-32H 

30 

16±  002 

8  2±0.15 

3  5±0  08 

.19±  009 

6  6±0  31 

7.1±0.50 

13  3±1.08 

19  9±1  06 

25±1  6 

34 

33^-35 

30 

17±  002 

7  6±0  12 

3  0±0  06 

14±  005 

5.2±0  37 

3.0±0  46 

5  0±0  86 

10  2±1  04 

35±3.0 

37 

36-38 

30 

.16±.002 

7  3±0  14 

2  3±0  04 

1 1 ±  003 

0  4±0  04 

5.9±0.76 

1  0±0.14 

1  4±0.15 

1±0  3 

*The  larger  fluctuations  with  the  lower  temperatures  were  due  to  the  stoppage  of  the  cooling  apparatus 
for  a  short  time. 

SWEET-ORANGE  SEEDLINGS 
This  experiment  was  set  up  with  one-month-old  sweet-orange  plants 
in  a  manner  similar  to  that  described  for  the  preceding  experiments. 
In  this  case,  however,  all  cotyledons  were  removed  for  the  purpose  of 
equalizing  the  individual  supplies  of  stored  food.  Owing  to  a  shortage 
of  suitable  plant  material  only  four  sets  of  30  plants  each  were 
employed.  The  temperatures  were  13°,  24°,  31°,  and  37°  C.  After 
two  weeks  the  plants  were  examined  and  measurements  of  the  growth 
were  made  (table  4). 


TABLE  4 

Growth  of  Sweet-orange  Seedlings  at  Different  Temperatures  for  a 

Two-weeks'  Period 


0 

CO 

c 
ca 
3. 

"o 
u 

V 

s 

3 

Stem 

Leaves 

Tap 

root 
elonga- 
tion 

Lateral  roots 

Total 
root 
elonga- 
tion 

Root  hairs 

3 
*h 

a 

a 

H 

Diam- 
eter 

Height 

Num- 
ber 

Fresh 
weight 

Num- 
ber 

Elonga- 
tion 

"8, 

—    - 

=  4 

a  — 
2 

o  0> 

"  C 

rn. 

cm. 

gm. 

cm. 

<•  m . 

c  in . 

13 

30 

.16±  002 

6.5±0  12 

2.1±0  02 

08±005 

O.liO.Ol 

0  0 

0  0 

0  1±0  01 

11 

7±3 

24 

30 

Hi  i    mil 

6.7±0.18 

2.7±0.09 

09±  005 

2  8±0.21 

1.3±0  28 

1  6±0  39 

4.4±0.49 

29 

25±3 

31 

30 

.15±.002 

6.6±0.18 

2  9±0  06 

09± .  006 

0  l±.0O5 

0  1±0  03 

3  4±0  36 

24 

60±5 

37 

30 

15±.002 

6.4±0.17 

2  4±0  06 

06±  004 

0.4±0  06 

0  1±0  03 

O.lrfcO  01 

0  5±0.06 

20 

8±2 

*  Many  of  the  plants  at  the  extreme  temperatures  produced  no  root  elongation  during  the  experi- 
ment and  were  thus  automatically  eliminated  from  the  root-hair  study. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment 


89 


80 


70 


60 


^so\ 

s3Ks 


^ 


n!* 


20 


10 


/O           /5           20          25           30     ,      15  *0 
Degrees    Cent/grade >> 

Fig.  2.     Graphs  showing  root-hair  production  with  citrus  seedlings  grown 
at  various  maintained  temperatures. 

The  curves  for  the  sweet-orange  plants  (figs.  1  and.  2)  follow  a 
course  similar  to  that  described  for  the  grapefruit  and  the  sour-orange 
plants.  The  optimum  temperature  for  root-hair  production  appears 
to  be  somewhat  higher  than  that  for  total  root  elongation.  A  study  of 
the  tabulated  data  for  this  experiment  furthermore  suggests  that  the 
top  development,  as  evidenced  by  the  number  and  fresh  weight  of  the 
leaves  produced,  was  influenced  to  a  limited  extent  by  the  temperature, 
even  though  the  experimental  period  was  short. 


DISCUSSION 
The  preceding  experiments  indicate  that  there  exists  a  minimum, 
an  optimum,  and  a  maximum  temperature  for  root  elongation  and  the 
production  of  root  hairs.   The  minimum  temperature  under  the  experi- 


90  University  of  California,  Publications  in  Agricultural  Sciences      [Vol.  5 

mental  conditions  was  roughly  12°,  the  optimum  26°,  and  the  maxi- 
mum 37°  C.  The  fact  that  the  apparent  optimum  for  the  sweet-orange 
plants  was  about  two  degrees  lower  than  the  average  value  stated,  is 
probably  due  to  the  greater  temperature  intervals  used  in  that  experi- 
ment. Also,  the  somewhat  lower  maximum  temperature  apparent  for 
the  grapefruit  seedlings  may  be  explained  upon  the  basis  of  insufficient 
illumination,  resulting  in  a  low  photosynthetic  activity  which  was 
here  coupled  with  a  high  respiratory  rate.  The  average  temperature 
values  are  in  accord  with  those  found  by  Peltier  (1920)  for  the  top 
growth  of  grapefruit  plants.  Using  five-degree  temperature  intervals 
Peltier  found  15°  C  for  the  minimum,  20  to  30°  for  the  optimum,  and 
35°  C  for  the  maximum  temperatures. 

It  is  of  interest  to  note  that  the  optimum  temperature  did  not  lie 
halfway  between  the  minimum  and  maximum  temperatures,  but 
rather  closer  to  the  maximum  end  of  the  range.  This  is  especially 
noticeable  in  the  case  of  the  temperatures  found  most  favorable  for 
root-hair  production,  the  apparent  optima  in  this  case  being  slightly 
higher  than  those  obtained  for  root  elongation. 

Although  the  periods  used  for  experimentation  were  short,  some 
responses  were  obtained  with  the  slower  growing  tops.  The  number 
and  fresh  wTeight  of  the  leaves  showed  a  behavior  similar  to  that  of  the 
roots  in  relation  to  temperature.  The  plants  maintained  at  moderate 
temperatures  were  able  to  start  the  development  of  a  second  pair  of 
leaves,  but  those  exposed  to  the  extreme  temperatures  were  unable 
to  do  so. 

Finally  it  may  be  observed  that  the  temperature  coefficient  is  of 
considerable  magnitude  for  the  different  phases  of  root  growth  at  the 
lower  temperatures,  but  decreases  rapidly  with  increased  temperatures 
and  approaches  zero  at  the  highest  temperatures  (Pawcett,  1921). 


HYDROGEN-ION  CONCENTRATIONS 
The  influence  of  the  reaction  of  the  culture  solution  on  root  growth 
was  studied  in  several  series  of  cultures  of  sour-orange  seedlings.  The 
original  reaction  of  the  nutrient  solution  (pH  6.0)  was  changed  by 
adding  hydrochloric  acid  or  sodium  hydroxide.  The  colorimetric 
method  was  employed  for  the  determination  of  the  pll  value  of  all 
solutions. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  b.y  Environment 


91 


SOLUTIONS  AT  pH  4.0  TO  9.0  WITH  DAILY  ADJUSTMENTS  OF  THE  pH 
(PEELIMINAEY  EXPEEIMENT) 

Four  sour-orange  plants  were  grown  in  each  of  six  2-quart  jars 
which  contained  solutions  adjusted  to  the  following  pH  values : 
4,  5,  6,  7,  8,  and  9.  Daily  pH  determinations  were  made  with  all 
solutions  and  readjustments  were  made  where  necessary.  It  was 
observed  that  the  plants  acted  upon  the  solutions  in  such  a  way  as  to 
change  the  pH  toward  6.  The  daily  change  in  the  acid  solution  was 
slight,  but  that  in  the  neutral  and  the  alkaline  solutions  was  con- 
siderable, amounting  in  one  case  to  0.9  pH.  Undoubtedly  the  excretion 
of  carbon  dioxide  by  the  plant  roots  was  largely  responsible  for  the 
changes  observed.  After  three  weeks'  growth,  a  peculiar  thickening 
was  noticed  in  the  ends  of  many  of  the  roots  growing  in  the  solution 
at  pH  4.  Sections  were  made  of  these  abnormal  portions  and  it  was 
found  that  the  increased  size  was  due  to  a  considerable  increase  in  the 
cortical  tissue.  A  cross-section  taken  through  the  thickened  region 
of  one  of  these  roots  is  represented  by  the  drawing  in  figure  3.  It 
may  be  observed  that  the  central  tissues  of  the  root,  particularly  the 
xylem  and  the  phloem,  were  still  in  the  primary  state  characteristic 
of  a  very  young  and  healthy  root.  The  cortical  layer,  however,  was 
much  thicker  than  that  of  a  normal  root  and  occupied  the  greater  por- 
tion of  the  cross-sectional  area.  A  cross-section  of  a  normal  root  of 
the  same  species  is  represented  for  comparison  in  figure  4. 


A  B 

Fig.  3.  (A)  Diagram  of  a  cross-section  of  a  sour-orange  root  showing 
relative  development  of  cortex  and  stele  from  a  culture  solution  having  a 
reaction  of  pH  4.0. 

(B)  Diagram  of  a  cross-section  of  a  normal  sour-orange  root,  a  —  cortex; 
b  =  phloem;  c  =  pith;  d  =  xylem;  e  =  perieycle. 


92 


University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 


Fig.  4.     Graphs  showing  root  growth  and  root-hair  production  with  sour-oranf 
seedlings  grown  in  solution  ;it   various  hydrogen-ion  concentrations. 


GROWTH  IN  SOLUTIONS  AT  pH  5.0  TO  9.0  WITH  DAILY  ADJUSTMENTS 

OF  THE  pH 

An  experiment  with  six-weeks-old  plants  was  set  up  similarly  to 
the  one  just  described,  using  five  cultures  of  30  plants  each,  in  glass 
jars  holding  three  and  one-half  liters  of  solution.  The  solutions  were 
adjusted  to  the  values:  pH  5.0,  6.0,  7.0,  8.0,  and  9.0.  It  was  observed 
that  light  colored  precipitates  were  formed  in  the  solutions  of  pH  8.0 
and  9.0.  These  precipitates  were  probably  largely  composed  of 
calcium  and  iron  phosphates,  as  these  substances  are  relatively 
insoluble  in  alkaline  solutions.  The  cultures  were  maintained  at 
29  ±  1°  C  and  received  continuous  illumination  from  a  500-watt  light 
suspended  about  one-half  meter  above  the  plants. 

An  examination  of  the  plants  after  a  growth  period  of  two  weeks 
brought  out  the  following  facts.  In  general,  the  development  of  the 
root  systems  was  fairly  uniform  over  the  entire  pll  range.     The  most 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment  93 

abundant  production  of  root  hairs,  however,  occurred  at  pH  5.0  and 
6.0,  viz.,  33  ±  3  and  28  ±  3,  respectively.  The  smallest  number 
(11  ±  3)  was  produced  in  the  most  alkaline  solution  (pH  9.0). 
Owing  to  the  occurrence  of  large  daily  variations  (0.5  to  0.8  pH)  in 
the  alkaline  solutions,  it  is  evident  that  much  of  the  growth  was  made 
in  solutions  closer  to  neutrality  than  the  initial  pH  values  indicate. 


SOLUTIONS  AT  pH  4.0  TO  9.0  WITH  pH  ADJUSTMENTS  MADE  TWICE 

DAILY 

This  experiment  consisted  of  a  repetition  of  the  preceding  one  with 
a  few  modifications.  The  extent  of  the  pH  range  was  increased  to 
include  a  more  acid  solution,  i.e.,  pH  4.0.  Because  of  the  rapidity  with 
which  the  alkaline  solutions  underwent  change,  it  seemed  desirable  to 
adjust  the  hydrogen-ion  concentrations  twice  each  clay.  These  adjust- 
ments were  made  at  9  a.m.  and  5  p.m. 

The  plant  material  employed  in  this  experiment  consisted  of  seven- 
weeks'  old  sour-orange  seedlings.  These  plants  were  carefully  selected 
in  five  lots  each  in  order  to  obtain  comparable  populations  for  the 
different  treatments.  Continuous  illumination  from  a  500-watt  light 
was  again  used  but  the  temperature  was  reduced  to  25  ±  1°  C.  The 
experiment  extended  over  a  period  of  10  days. 

The  data  in  table  5  show  that  the  maximum  growth  of  roots 
occurred  in  solutions  having  reactions  of  pH  6.5.  The  production  of 
root  hairs  also  shows  a  correlation  with  the  hydrogen-ion  concentra- 
tion. Prom  the  graph  in  figure  5  it  may  be  seen  that  the  production 
of  root  hairs  Avas  strongly  depressed  at  pH  4.0,  rose  to  a  maximum  at 
pH  5.0,  and  declined  steadily  with  the  higher  pH  values.  The  optimum 
hydrogen-ion  concentration  for  the  production  of  root  hairs  was  con- 
siderably greater  than  that  for  root  elongation,  the  latter  being 
situated  near  the  point  of  neutrality. 


DISCUSSION 

The  condition  permitting  maximum  root  growth  was  found  to  exist 
in  the  region  of  neutrality.  The  most  favorable  range  for  root-hair 
production,  however,  occurred  in  a  distinctly  acid  solution  (pH  5.0). 
Both  the  production  of  root  hairs  and  the  total  root  elongation  showed 
evidence  of  depression  in  the  most  acid  (pH  4.0)  and  the  most  alkaline 
solutions  (pH  9.0). 


94 


University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 


W 
PQ 

Eh 


Eh 
55 
« 
W 
55 

o 
o 

o 

55 

H 


a 
K 

E-i 
55 

w 
si 
w 


5; 

g 

o 

02 


Q 
02 


55 

< 
as 
o 

CS 
& 

o 

02 


o 
2 


01 

'3 
o 

Q 
« 

%  w  * 

a£.S 
< 

CO    f     cm    ao    *<*• 

o   cm   ~*  o   © 

-H  -H  -H  -H  -H 

— >    —    CO    •«*<    CS 

3  3.2 

Zog 

- .       —       ."        "-       r    - 

CM     CM     CM    CM    CM 

o_o 
E"1  "3 

**i     CO     ©     t1-    ^»« 

-     *     O    W    N 

-    O    O    O    ©    © 

S  -H  -H  -H  -H  -H 

*■*    ©    CO    O    O    CC 

©     CO     ^     CO     *■* 

o 

o 

"3 

u 
m 

CS 

M  C 
C   O 

o-- 

s 

»    ^     W     M     N 
©     CO     *0     CO     <-• 

■     ©©©©© 

S  -H  -H  -H  -H  -H 

«    UJ     O     tO    CO     C5 
©     CM     CO     •"-■     © 

u 

2 

3 

©    CO    ©    CO    "5 
CO     CO     V}     CO     CM 

©     ©     ©     ©     © 

■H  -H  -H  -H  -H 

O0     <D    —     N     ifl 
»H     N    ^f     «     H 

o  2 

o  3 

v 

CO     ©     CO     CM     CM 
-    O    ©    ©    ©    © 

£  -H  -H  -H  -W  -H 

U     "**«     CO     CO     CM     © 

©      —      — <      -H      © 

> 

■5  2 

•r    m    f    -v   W 
©   ©   ©  o  o 
©  ©  ©  ©  © 

i  jjij  jj  jj  jj 

S 

3 

CM     W     CO     CO     — 
©     ©     O     ©     © 

©     ©     ©     ©     © 

-H  -H  -H  -H  -H 

■—•     "*f"     CM     CM     — i 
CM     CM     CM     CM     CM 

S 

'S3 

W 

OJ     CM     ©     CM     i-t 

O    ~    <-t    *H    ^ 

.©©©©© 

6  -H  -H'-H  -H  -H 

«  c»  ©  o>  ©  © 

N    CO     N    M     N 

0) 

i 

OS 

3 

CM     CM     »-•     CM     CM 
©     ©     O     ©     © 
©    O    ©    ©    © 

E  -H  -H  -H  -H  -H 

«     ffl    N    N    h.     ffl 

E- 

©     ©     ©     ©     © 

co   co   co  co  eo 

W 

a 
a 

c 
_o 

.3 

'hi 

a 
> 

H    O    ^    W    ifl 
©    o   ©   ©   © 

1        M  J, 

©         o   ©    © 

< 

co   co   ©   »o   *n 

TT°TT 

©  ©        ©  © 

©    ©    uO    ©    © 
V   U5   to   oo   o> 

1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment  95 

The  fact  that  comparatively  few  hairs  were  produced  in  the 
alkaline  solutions  (pH  8.0  and  9.0)  of  the  preceding  experiment  is 
worthy  of  note  in  another  connection.  It  is  a  common  belief  that 
Citrus  plants  do  not  produce  root  hairs.  At  the  same  time  it  may  be 
noted  that  the  soils  common  to  the  Citrus  regions  of  southern  Cali- 
fornia have  a  decided  alkaline  reaction.  Therefore  it  seems  that  the 
belief  in  the  non-production  of  root  hairs  by  Citrus  roots  may  be 
related  to  the  characteristic  alkaline  reaction  of  the  soil  solution  which 
acts  to  suppress  their  formation.  This  explanation  appears  to  be  a 
logical  one  and  worthy  of  further  experimental  study. 

OXYGEN 

Three  similar  lots  of  30  plants  each  were  placed  in  2-quart  jars 
fitted  with  cork  stoppers,  each  jar  containing  10  plants.  Three  of  the 
jars  were  filled  with  nutrient  solution  which  had  been  previously 
aerated  for  three  hours.  These  solutions  were  also  continuously 
aerated  during  the  period  of  the  experiment.  A  second  lot  of  plants 
grew  in  an  unaerated  solution,  which  contained  initially  a  considerable 
amount  of  dissolved  oxygen.  The  third  lot  of  plants  was  placed  in 
jars  containing  a  solution  which  had  been  boiled  in  a  partial  vacuum 
to  drive  out  the  dissolved  air.  All  jars  were  sealed  with  a  paraffin- 
petrolatum  mixture  (80%  paraffin,  20%  petrolatum),  but  the  jars  of 
the  aerated  and  control  cultures  were  provided  with  vents  so  that  the 
outside  air  had  access  to  the  solutions.  The  jars  containing  the  evacu- 
ated solution  were  equipped  with  soda-lime  tubes  for  the  purpose  of 
preventing  the  accumulation  of  carbon  dioxide  in  high  concentrations. 

Continuous  aeration  was  secured  by  the  use  of  a  water  pump 
described  by  Allison  (1922).  The  pump  was  connected  with  three 
glass  tubes,  each  extending  into  the  solution  of  one  of  the  aerated 
cultures.  Each  tube  was  26  centimeters  long  and  was  provided  with 
a  small  perforated  bulb  at  the  lower  end.  This  allowed  the  air  to  be 
forced  down  into  the  solution  to  a  depth  of  about  20  centimeters  and 
expelled  in  the  form  of  small  bubbles. 

Analyses  for  dissolved  oxygen  were  made  of  the  culture  solutions 
at  the  beginning  and  end  of  each  experiment.  The  method  used  was 
that  devised  by  Winkler  and  described  by  Treadwell  and  Hall  (1915). 
The  technique  given  by  Allison  and  Shive  (1923a)  for  applying  this 
method  to  small  quantities  of  solution  was  followed.  Some  disparity 
was  observed  in  the  actual  amounts  of  dissolved  oxygen  found  in  the 
different  experiments.    This  lack  of  agreement  may  have  been  due  to 


96  University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 

the  temperature  differences  and  to  possible  inaccuracies  in  the  N  '25 
sodium  thiosulfate  solution  required  for  titrating  the  iodine  liberated 
by  oxidation.  In  order  to  correct  this  situation  the  analyses  in  the 
third  experiment  were  conducted  at  the  temperature  at  which  growth 
took  place,  and  a  freshly  prepared  N/25  thiosulfate  solution  was 
used.  It  is  felt  that  this  latter  set  of  analyses  is  reasonably  trust- 
worthy and  representative. 

AERATION   CONDUCTED   AT    27  ±  1.5°  C 

Two-months-old  plants  were  employed  in  an  experiment  extending 
over  a  period  of  12  days.  Illumination  was  supplied  continuously  by 
a  500-watt  light  suspended  about  one-half  meter  above  the  plants. 
On  the  sixth  day  the  soda-lime  associated  with  the  boiled-solution 
cultures  was  removed.  The  experimental  results  are  described  briefly 
in  the  following  paragraphs. 

The  analyses  of  the  original  solutions  showed  that  the  preliminary 
aeration  of  the  culture  solution  had  little  effect  in  increasing  its  oxygen 
content.  Apparently  the  pouring  and  shaking  attendant  to  the  pre- 
paration resulted  in  the  incorporation  of  nearly  sufficient  oxygen  to 
saturate  the  solution.  The  treatment  of  combined  boiling  and  evacua- 
tion, however,  decreased  the  oxygen  content  considerably,  although 
there  was  still  a  significant  amount  of  dissolved  oxygen  left  in  the 
solution. 

The  results  of  the  analyses  at  the  end  of  the  experimental  period 
reveal  two  interesting  points.  First,  the  oxygen  content  of  the  boiled 
solution  remained  practically  constant.  It  is  certain  that  the  outer 
air  had  access  to  these  solutions  in  spite  of  the  seals  used,  and  that 
withdrawal  of  oxygen  from  the  solutions  by  the  roots  was  coupled 
with  an  equally  rapid  absorption  of  oxygen  by  the  solutions  from  the 
air.  Second,  the  oxygen  content  of  the  control  culture  solutions  had 
been  eventually  reduced  to  that  of  the  boiled  solutions,  apparently 
by  the  action  of  the  plant  roots. 

No  significant  differences  in  plant  growth  were  evident  between  the 
control  and  the  boiled-solution  cultures.  Since  the  original  differences 
in  oxygen  content  were  not  maintained,  it  is  probable  that  the  amounts 
of  dissolved  oxygen  in  the  two  series  of  solutions  were  very  similar 
over  the  major  part  of  the  experiment.  An  unfortunate  fungus 
infestation  retarded  the  growth  of  the  aerated  plants.  For  this  reason 
all  plants  receiving  aeration  had  to  be  discarded  at  the  end  of  the 
experiment. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment  97 


H    > 


M 

n 

> 

2. 

0 

p 

b 

a. 

3 

a. 

H 

-i 

n 

» 

g 

£ 

CO    CO                     Cn 

en                   Cn 

Cn 

cr 

Oi     ^                     •— 

to                   CO 

cn 

IK 

rf*    OO                     OC 

to 

CO                     Oi 

CO 

cn 

rt 

O 

X 

<< 

Cn 

to 

5" 

TO 
» 

•a 

CO 

OS 

s 

^j 

(R 

Cfl    *    *             rf* 

Cn    *»■            OS 

33    O 

> 

>—    ^j    os             —1 

©00            oo 

CO    CO 

"I 

*-•    o    •—          © 

^    Oi            CO 

^.    rf*. 

3 

CO 

D 

ST 

*. 

*- 

cn 

-i 

30 

55 

o 

2,2 

•o  c 

5*1 

CO 

© 

=> 

p  o 

g 

^1 

*-i 

o 

p 

ff 

f 

f 

a 

3 

o 

© 

© 

o 

to 

© 

CO 

CO 

co 

.J 

3 

>— 

ffi 

1W* 

CC 

to 

Cn 

« 

if 

f 

3 

o 

© 

© 

ET 

H- 

CO 

E 

o 

*. 

#» 

^. 

Z    ■ 

Cn 

cn 

c 

H- 

f 

f 

3 

o 

© 

© 

c 

_ 

© 

© 

tv 

(6 

o 

-J 

P 

< 

n 

o 

CO 

CO 

H- 

f 

f 

3 

o 

© 

© 

£§* 

CO 

CO 

o 

rc     , 

to 

Cn 

o£ 

to 

£». 

_ 

0,5 

p    -, 

£ 

f 

3 

o 

© 

© 

ET.  O 

to 

S3 

~-I 

°  S- 

—J 

Cn 

to 

co 

X 

2 

CO 

00 

C 

ff 

f 

3 

^ 

o 

© 

© 

o- 

P 

CO 

*» 

CO 

<d 

n 

CO 

OS 

«k 

p 

C71 

~4 

PI 

O   3 

c 
o 

H- 

f 

f 

2 

EB 

o 

© 

3  TO 
p 

o 

© 

CO 

** 

CO 

^J 

o 

&S 

it* 

^3 

H- 

f 

f 

3 

© 

© 

© 

oo 

p 

to 

C   o 

en 

© 

P3 

~.o 

to 

™  o 

4- 

4- 

o 

© 

to 

2  cr 

© 

00 

M 

H 


s 

O 

*g 

03 

O 

s» 
6 

► 
2; 

S3 


CO 


W 
F 


98  University  of  California  Publications  in  Agricultural  Sciences      [Vol.5 


AEKATION  CONDUCTED   AT   25  ±  1°  C 

In  an  experiment  using  the  methods  just  described,  two-months-old 
sour-orange  plants  were  grown  for  about  three  weeks  (table  6). 

In  the  control  solutions  the  original  oxygen  content  was  very  close 
to  that  of  the  solutions  aerated  for  three  hours,  as  noted  in  the 
previous  experiment,  showing  that  a  high  degree  of  aeration  was 
secured  during  the  course  of  preparation.  During  the  course  of  the 
experiment  the  action  of  the  roots  upon  the  control  solution  eventually 
reduced  its  oxygen  content  to  that  of  the  boiled  solution,  which  had 
been  increased  by  the  absorption  of  atmospheric  oxygen. 

Colorimetric  determinations  were  made  of  all  solutions  at  the 
close  of  the  experiment  in  order  to  determine  the  hydrogen-ion  concen- 
trations. Those  of  the  aerated  solutions  were  pH  6.4,  6.5,  and  6.6, 
those  of  the  control  solutions  6.3,  6.4,  6.3,  and  those  of  the  boiled 
solutions  6.3,  6.3,  6.3.  The  slightly  higher  values  for  the  aerated  cul- 
tures suggest  that  aeration  had  removed  some  dissolved  carbon  dioxide 
from  these  solutions. 

Table  6  shows  that  the  plants  of  the  control  and  boiled-solution 
cultures  were  characterized  by  a  very  similar  development.  As  no 
differences  greater  than  three  times  the  probable  error  are  evident,  it 
may  be  concluded  that  no  significant  differences  existed.  In  the  case 
of  the  aerated  cultures,  however,  the  increase  of  tap-root  elongation 
may  be  of  significance ;  in  fact  the  longer  tap-root  development 
attained  by  the  aerated  cultures  was  obvious  when  the  plants  were 
examined. 

The  greater  relative  number  of  root  hairs  produced  in  the  aerated 
cultures  is  very  evident  from  the  data  presented  in  this  table.  It 
seems  that  root-hair  production  is  much  more  sensitive  to  aeration 
than  is  the  elongation  of  the  tap  or  lateral  roots. 

Table  7  gives  the  results  of  experiments  in  which  the  boiled-solution 
cultures  were  omitted.  The  presence  of  fungi  upon  the  roots  of 
plants  in  the  aerated  cultures  suggested  to  the  writer  that  fungus 
spores  were  being  carried  into  the  aerated  solutions  by  way  of  the  air 
stream.  In  order  to  remedy  this  trouble  a  loose  cotton  filter  and  a 
wash  bottle  containing  distilled  water  were  inserted  in  the  air  line 
between  the  pump  and  the  aerated  cultures.  This  device  proved  to 
be  effective,  as  all  aerated-culture  plants  subsequently  remained 
entirely  free  from  fungus  attack. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment 


99 


a 

> 

o 

B 

3 

3, 

0 

1 

H 

"I 

a> 

p 

3 

a> 

3 

(n    0<        Oi 

in 

o« 

tr 

■^1    Ot        -^J 

*q 

H-     CO           OS 

3s 

■^-8 

O 

X 

<< 

as 

5' 
to 

TO 

a> 

3 

CO 

•J\ 

a> 

^ 

4~ 

> 

1 

CO 

LO 

H-           H- 

f^ 

a 
a 

» 

o 

S 

*i 

o 

p 

Oi 

2,2 

CO 

:o 

•2-3 

o 

c- 

S  ST 

r+    fC 

CD    1 

0 

OS 

33 

o 

p 

ff        ff 

3 

3 

«i 

o 

- 

o 

s 

(D 

to 

■o 

-1 

GO 

3 

-a 

■a 

X 

CO                   Oo 

ff        ff 

cs 

3 

2. 

TO' 

o 

- 

S" 

_ 

l"- 

CO                       CO 

3 

to 

'O 

C 

H-            H- 

3 

o              o 

tr 

^ 

t-i 

o 

r 

P 

< 

a 

CO                           +• 

ff        ff 

«3 

35" 
Sis 

CD 

o              o 

sfp* 

o              o 

CO 

<&  , 

to                     4»- 

ff        ff 

3 

in'O 

p  *i 

o              o 

?.  o 

ft                       CO 

CO                     to 

CO                          CO 

z 

o              to 

c 

H-            H- 

3 

t-1 

p 

o             o 

<r 

Cn                   4k. 

n 

CD 
•1 

O                     -J 

P 

OO                     tfk. 
CO                       •*»• 

n 

-.  5" 

O   3 

O 
O 

H-           tt- 

3 

co 

o 

3  TO 

P 

-a                o 

to 

ffc. 

,_. 

O  O 

—                           OO 

CO                       Ol 

3  s 

H-           H- 

3 

to  £. 
p 

o 

?-o 

-^1 

O   o 

o 

JO 

a  S- 

3     > 

OS                     OS 

ff         ff 

Q-  i    P  Si 

o                to 

<T>  3^  o 
X  P  n   X 

OO 

o 

jO 

S" 

*•" 

o 

55 

K 


o 

c! 

? 
6 

> 

o 
w 

oc 

H 
w 
o 


100  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  5 

The  data  presented  in  table  7  show  that  both  the  tap-root  and  the 
lateral-root  elongation  were  increased  by  aeration  of  the  culture 
solution.  The  production  of  root  hairs  was  greatly  increased  and 
appeared  to  be  the  most  marked  result  of  aeration. 

Hydrogen-ion  determinations  were  made  of  all  solutions  at  the 
close  of  the  experiment.  The  values  obtained  were  the  following: 
aerated  cultures,  pH  6.3,  6.3,  and  6.2 ;  control  cultures,  6.1,  6.1,  and  6.1. 

DISCUSSION 

In  general  the  preceding  experiments  have  brought  out  the  follow- 
ing facts.  In  the  preparation  of  the  nutrient  solution,  a  certain 
quantity  of  oxygen  was  introduced  from  the  air  (5.65  cc.  per  liter 
at  25°  C — table  7),  which  was  later  reduced,  presumably  by  the 
respiratory  action  of  the  plant  roots,  unless  replenished  by  aeration. 
This  quantity  of  dissolved  oxygen  closely  approaches  that  given  by 
Landolt,  Bornstein,  and  Roth  (1912)  for  complete  saturation,  viz., 
5.78  cubic  centimeters  per  liter  for  a  temperature  of  25°  C. 

The  development  of  the  plants  in  the  boiled-solution  cultures  dupli- 
cated that  of  the  control  cultures  within  the  limits  of  experimental 
error.  This  was  true  for  the  growth  of  all  organs  observed  and  sug- 
gests that  some  oxygen  was  being  absorbed  from  the  air  by  both  the 
boiled  and  the  control  solutions.  On  the  other  hand,  the  root  growth 
of  the  plants  in  the  aerated  solutions  showed  evidence  of  a  greater 
development  than  in  the  unaerated  solutions.  This  was  especially 
noticeable  in  the  case  of  root-hair  production. 

In  the  pH  of  the  aerated  solutions  there  was  seen  a  small  but 
constant  trend  toward  a  less  acid  reaction  than  in  the  pH  of  the  con- 
trol solutions.  This  suggests  the  presence  of  an  appreciable  amount  of 
dissolved  carbon  dioxide  in  the  unaerated  solutions  which  resulted  in 
the  formation  of  the  bicarbonate  ion  by  its  partial  dissociation.  There- 
fore, the  effect  of  the  aeration  treatment  appears  to  be  twofold:  first. 
the  oxygen  supply  was  constantly  renewed  ;  second,  the  carbon  dioxide 
excreted  by  the  roots  was  continually  driven  off  by  the  aerating 
process.  It  is  very  likely  that  both  conditions  operated  to  promote 
tin-  growth  of  the  plants.  Certainly  both  conditions  would  favor  the 
respiratory  activity  of  the  roots  and  increased  respiration  in  turn 
would  presumably  be  associated  with  increased  root  activity  and 
growth. 

The  responses  to  aeration  obtained  with  the  sour-orange  plants  are 
in  keeping  with  those  observed  for  many  other  plants.     For  example 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         101 

Stiles  and  Jorgensen  (1917)  found,  with  barley  and  balsam,  that  an 
aeration  of  the  culture  solutions  resulted  in  increased  yields.  Andrews 
and  Beals  (1919)  found  that  the  yield  of  maize  grown  in  water  cul- 
tures was  also  increased  by  aeration.  In  addition,  the  work  of 
Andrews  (1920),  Allison  (1922),  Allison  and  Shive  (1923),  and 
Knight  (1924)  has  shown  that  aeration  of  solution  cultures  was 
beneficial  for  various  plants  including  oats,  peas,  soy  beans,  wallflowers, 
and  mustard.  However,  not  all  plants  respond  to  aeration.  Free 
(1917)  reported  that  the  growth  of  buckwheat  plants  in  solution  cul- 
tures treated  with  air,  oxygen,  or  nitrogen  was  equal  to  that  obtained 
in  open  controls  or  in  sealed  cultures.  It  is  of  interest  to  note  that 
when  carbon  dioxide  was  passed  through  the  solutions  the  plants 
wilted  within  a  few  hours  and  died  within  a  few  days. 

A  few  investigators  have  also  noted  the  effect  of  oxygen  upon  the 
production  of  root  hairs.  Schwarz  (1883)  observed  that  a  relation 
existed  between  the  available  oxygen  supply  and  the  development  of 
root  hairs.  Miss  Snow  (1905)  found  that  the  root-hair  production 
of  corn  was  suppressed  when  the  roots  were  exposed  to  moist  air 
deprived  of  oxygen,  although  exposure  to  moist  air  was  ordinarily 
accompanied  by  an  abundant  production  of  root  hairs.  More  recently 
Bergman  (1920)  found  that  Impatiens  balsamina  produced  root  hairs 
in  aerated  water. 

MIXTUKES  OF  GASES 
In  order  to  determine  the  action  of  mixtures  of  gases  upon  the 
growth  of  Citrus  roots,  a  series  of  experiments  was  conducted  with 
sour-orange  seedlings  in  cultures  of  a  river  sand  of  medium  texture. 
In  the  earlier  experiments,  the  plants  were  grown  in  opaque  triple- 
necked  Woulff  bottles.  Two  small  seedlings  were  placed  in  each  of 
the  outer  necks  while  a  rubber  stopper  fitted  with  a  short  glass  tube 
was  inserted  in  the  center  neck.  Half  of  the  jars  (usually  six)  were 
those  having  tubulatures  at  the  base  (fig.  5).  These  tubulatures  were 
also  fitted  with  rubber  stoppers  containing  short  glass  tubes.  The 
jars  were  connected  to  a  delivery  tube  of  the  manifold  type,  which  in 
turn  was  connected  to  a  gas-holder  and  an  intervening  wash  bottle. 
The  gas  mixture  under  investigation  was  passed  through  each  of  the 
sand-filled  Woulff  bottles  daily  by  applying  a  slight  suction  to  the 
tube  extending  from  the  center  neck.  These  openings  were  kept  closed 
at  all  other  times  by  means  of  short  pieces  of  rubber  tubing  fitted 
with  clamps.  Control  cultures  in  a  similar  set  of  bottles  were  main- 
tained in  all  experiments   (fig.  6).     These  Woulff  bottles  lacked  the 


102 


University  of  California  Publications  in  Agricultural  Sciences       [Vol.  5 


tubulature  at  the  base,  and  the  center  neck  of  each  bottle  was  fitted 
with  a  long  and  a  short  glass  tube  instead  of  only  the  short  one  used 
for  the  treated  cultures.  Both  of  these  tubes  were  open  to  the  air 
so  that  a  normal  diffusion  of  gases  could  take  place  at  all  times.     The 


Figs.  5,  6,  and  7.  Diagrams  of  apparatus  used  for  experiments  on  the  effect 
of  gases.  No.  5,  Woulff  bottle  typo.  Side  view,  median  section.  No.  6,  Woulff 
bottle  type  as  used  in  control  cultures.  No.  7,  Straight  tube  type,  « =  glass 
stopper;  b  =  rubber  tubing;  c  =  wax  seal;  d  =  rubber  stopper;  e  =  glass  tubing; 
/=  glass  culture  tube;  g  =  sand  ;  h  =  glass  wool;  i  =  T-tube;  j  =  Woulff  bottle; 
k=  tubulature. 

sand  ill  all  cultures  was  moistened  with  nutrient  solution  at  the  begin- 
ning of  each  experiment  and  the  plants  were  customarily  sealed  in 
with  the  paraffin-petrolatum  mixture  in  order  to  render  the  system 
gas  tight. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         103 

Later  on  it  seemed  desirable  to  employ  a  container  in  which  a 
more  uniform  distribution  of  gas  could  be  assured,  as  there  was  some 
reason  to  believe  that  the  composition  of  the  gas  drawn  off  and 
analyzed  from  the  Woulff  bottles  was  not  identical  with  that  surround- 
ing the  plant  roots.  For  this  reason  several  of  the  experiments  were 
conducted  with  straight-walled  glass  tubes  having  an  average  diameter 
of  about  35  millimeters  and  a  length  of  30  centimeters.    These  cultures 


t^ 


Fig.  8.     Gasometer,  side  view,  median  section,     a  =  guide  for  inner  tank ;  b  =  gas 
outlet;   c  =  inner  tank;   d  =  gas;    e  =  water;   /  =  outer  tank. 


were  set  up  as  shown  in  figure  7,  using  two  plants  to  each  tube.  At 
the  time  of  planting,  the  cultures  were  irrigated  with  an  excess  of 
nutrient  solution,  the  surplus  solution  being  removed  by  applying  a 
slight  suction  to  the  lower  outlet  tube  and  drawing  several  hundred 
cubic  centimeters  of  air  through  each  tube.  The  lower  outlet  tube 
was  then  attached  to  the  gas  line  and  the  experimental  gas  mixture 
drawn  through  the  treated  cultures  once  or  twice  each  day. 

The  gas-holder  (fig.  8)  consisted  of  a  galvanized  iron  tank  inverted 
within  a  larger  tank  of  the  same  material.  The  outer  tank  was  par- 
tially filled  with  water  to  prevent  the  escape  of  gas  from  the  inverted 


104  University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 

one.  A  guide  of  vertical  metal  rods  was  inserted  in  the  space  between 
the  two  tanks  in  order  to  support  the  inner  and  movable  tank.  Gas 
could  be  run  into  or  withdrawn  from  the  gasometer  by  means  of  a 
small  galvanized  iron  pipe  projecting  through  the  bottom  of  the  outer 
tank  and  extending  above  the  water  level.  There  was  also  a  second 
outlet  at  the  top  of  the  inner  tank  which  was  closed  by  a  stopcock. 

Analyses  were  made  at  intervals  (usually  every  four  days)  of  the 
gas  drawn  off  from  the  treated  cultures  and  from  the  gasometer.  All 
such  analyses  were  made  with  a  portable  Haldane  apparatus  (Haldane, 
1920).  In  addition,  at  the  end  of  each  experiment,  a  set  of  analyses 
was  made  of  the  gas  from  the  control  cultures.  Plant  measurements 
including  both  root  and  top  growth  were  also  taken  at  this  time. 
Owing  to  the  presence  of  soil  incrustations  on  the  surface  of  the  roots 
and  to  the  likelihood  of  breakage  during  removal  from  the  soil,  no 
attempt  was  made  to  study  the  production  of  root  hairs  as  influenced 
by  the  various  gas  treatments. 


GKOWTH  IN  A  GAS  MIXTUKE  SIMILAE  TO  THAT  OF  NORMAL  AIR 

Two  experiments  were  conducted  to  test  the  plant  material  and 
the  experimental  conditions.  The  roots  of  paired  six-weeks-old  plants 
in  Woulff  bottles  were  treated  with  a  gas  mixture  similar  to  normal 
air  in  composition.  In  the  first  experiment  24  plants  were  used  both 
in  the  aerated  and  in  the  unaerated  series.  A  gas  mixture  consisting 
of  0.3  per  cent  carbon  dioxide,  20.0  per  cent  oxygen  and  79.7  per  cent 
nitrogen  was  passed  through  each  of  the  treated  cultures  daily.  A 
quantity  in  excess  of  the  pore  space  was  removed  from  each  culture. 
The  plants  were  maintained  at  a  temperature  of  27  ±  1°  C  under 
continuous  illumination  for  three  weeks.  At  the  end  of  the  experi- 
ment the  growth  of  roots  in  the  two  cases  showed  no  significant 
differences. 

A  second  experiment,  using  two  series  of  30  plants  each  in  culture 
tubes,  was  conducted  in  a  fashion  similar  to  the  previous  one.  How- 
ever, the  daily  renewals  of  gas  were  accomplished  by  withdrawing  a 
much  smaller  quantity  (usually  7.">  to  80  cc.)  from  each  culture.  The 
volume  of  gas  removed  was  approximately  three  times  the  pore  space 
occupied  by  the  soil  gas.  A  temperature  of  25  ±  1°  C  was  maintained 
for  the  l.'i  days  of  the  experiment. 

A  similar  development  was  again  noted  with  the  plants  of  the 
treated  and  control  series,  all  measured  differences  being  well  within 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         105 

the  limits  of  the  probable  errors.  It  should  be  stated  that  although 
the  gas  mixture  used  for  the  treatments  was  similar  to  air  in  composi- 
tion, the  gas  withdrawn  from  the  treated  cultures  contained  less 
oxygen  and  more  carbon  dioxide  than  did  the  gas  from  the  gasometer 
or  from  the  open  control  cultures.  For  example,  the  gas  analyzed 
from  the  treated  tube  cultures  had  an  average  composition  of  2.1 
per  cent  carbon  dioxide,  16.6  per  cent  oxygen,  and  81.1  per  cent 
nitrogen  as  contrasted  to  that  withdrawn  from  the  control  cultures 
which  was  0.6  per  cent  carbon  dioxide,  20.5  per  cent  oxygen,  and  78.9 
per  cent  nitrogen.  This  change  in  composition  was  undoubtedly  asso- 
ciated with  the  respiratory  activity  of  the  plant  roots  which  resulted 
in  an  accumulation  of  carbon  dioxide  and  a  disappearance  of  oxygen 
in  the  treated  cultures.  A  similar  condition  was  precluded  in  the  open 
control  cultures  by  the  continuous  gaseous  interchange  with  the 
outside  air. 


GROWTH  IN  MIXTURES  OF  REDUCED  OXYGEN  AND  INCREASED 

NITROGEN 

Two  experiments  were  conducted  with  Woulff  bottles  as  follows : 
In  the  first  experiment,  a  gas  mixture  with  a  very  low  oxygen  content 
(1.2%)  was  administered  to  the  roots  of  seven-weeks-old  plants.  These 
plants  were  grown  for  17  days  under  the  usual  artificial  illumination 
and  at  a  temperature  of  28  ±  1°  C.  Instead  of  the  paraffin-petrolatum 
mixture,  a  soft  modelling  clay,  '  plasteline, '  was  used  for  sealing  in 
the  plants.  Analyses  of  the  soil  gas  are  reported  in  table  8  and  the 
growth  of  roots  in  table  9. 

TABLE   8 
Composition  of  the  Soil  Gas  in  Woulff-bottle  Cultures  with  Reduced 

Oxygen  Supply 


Culture 

Day  of 

experimental 

period 

Number 

of 
analyses 

Per  cent 

co2 

Per  cent 
O2 

Per  cent 

N2 

Gas-treated 

' 

3 
6 

9 
13 

,           16 

6 
6 
6 
6 
6 

1  2±0.11 
1  0±0.03 
1  2±0.03 
0  9±0.02 
0.8±0  01 

»4.0±0.33 
1  5±0.29 
0  8±0  17 
0  6±0.09 
0  6±0  13 

94.8±0.32 
97.5±0.30 
98.0±0.17 
98.5±0.09 
98.6±0.14 

1.0=h0  03 

1.5±0  18 

97.5±0.20 

Control 

16 

6 

8 

0  6±0  08 
0  2±0.06 

20.4±0.09 
1.2±0.09 

79.0±0.03 
98  6±0.12 

*  The  high  oxygen  content  may  have  been  due  to  some  residual  oxygen  in  the  cultures  during  the 
first  few  days  of  the  experimental  period. 


106 


University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 


TABLE  9 
Growth  op  Sour-orange  Plants  in  Woulff -bottle  Cultures  with  Low 

Oxygen  Content* 
Temperature  28  ±  1°  C 


Num- 
ber of 
plants 

Stem 

Leaves 

Tap 

root 
elonga- 
tion 

Lateral  roots 

Total 

Culture 

Diam- 
eter 

Height 

Num- 
ber 

Fresh 
weight 

Num- 
ber 

Elonga- 
tion 

root 
elonga- 
tion 

Gas-treated 
Control 

24 
22 

cm. 
14±  002 
14±  002 

cm. 
6  7±0  14 
7.6±0  22 

2  2±0.04 

3  2±0  12 

gm. 
0  09±  005 
0  10±006 

cm. 

0  0 

2.3±0.35 

0  0 
5.9±0.63 

cm. 

0  0 

6  4±0.87 

cm. 

0  0 

8  7±0  94 

*  See  table  8  for  experimental  conditions. 

It  will  be  seen  from  table  9  that  the  root  elongation  of  the  treated 
plants  was  entirely  cheeked.  Since  the  carbon  dioxide  percentages  of 
the  soil  atmosphere  of  the  treated  and  the  control  cultures  were  of  the 
same  magnitude,  it  seems  highly  probable  that  the  suppression  of  root 
elongation  with  the  treated  plants  was  due  to  the  deficiency  of  oxygen 
in  the  gas  enveloping  the  plant  roots. 

In  a  second  experiment  with  Woulff -bottle  cultures,  a  gas  mixture 
of  a  high  oxygen  content  (8%)  was  administered  to  the  roots  of  five- 
weeks-old  plants  with  the  temperature  maintained  at  30  ±  1°  C.  In 
order  to  diminish  quickly  the  oxygen  content  of  the  treated  cultures, 
a  gas  containing  only  1  per  cent  of  oxygen  was  used  for  the  first  two 
days,  after  which  a  second  mixture  containing  about  8  per  cent  of 
oxygen  was  employed  for  13  days  more.  This  resulted  in  a  low  initial 
oxygen  content  of  the  treated  jars  (table  10),  but  subsequent  analyses 
showed  a  relatively  constant  average  oxygen  content  of  about  7 
per  cent. 

TABLE  10 
Composition  of  the  Soil  Gas  in  Woulff-bottle  Cultures  with  Reduced 

Oxygen  Supply' 


Culture 

Day  of 

experimental 

period 

Number 

of 
analyses 

Per  eent 
COa 

Per  cent 

o2 

Per  cent 
N« 

Gus-treated 

2 
5 
9 
12 

k           15 

7 

7 
7 
7 
7 

0  8±0  03 

1  l±0  02 
1  2±0.02 
1  3±0  03 
1.2±0.03 

2  6±0  43 
6.6±0  04 
7  2±0  06 

6  9±0.05 

7  0±0  14 

96  6±0  45 
92  3±0  05 
91.6±0  07 
91  8±0  05 
91.8±0  12 

Average 

1   ldbO  02 

6  1±0  21 

92  8±0  23 

15 

7 
6 

0  6±0  07 
0.5±0  08 

20  4±0  05 
7.9±0.52 

79  0±0  03 

91  6±0.50 

1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         107 

TABLE  11 
Growth  of  Sour-orange  Plants  in  Woulff-bottle  Cultures  with  Low 

Oxygen  Content* 
Temperature    30  ±  1°  C 


Num- 
ber of 
plants 

Stem 

Leaves 

Tap 

root 
elonga- 
tion 

Lateral  roots 

Total 

Culture 

Diam- 
eter 

Height 

Num- 
ber 

Fresh 
weight 

Num- 
ber 

Elonga- 
tion 

root 
elonga- 
tion 

Gas-treated 

28 
28 

cm. 
17±002 
17±  002 

cm. 
7.6±0  07 
7.9±0  09 

2.6±0.14 
3.4±0.11 

gm. 
09±  003 
11±  003 

cm. 
0  8±0  17 
2  4±0.36 

3.5±0  55 
7.4±0  40 

cm. 
2  0±0.48 
7.4±0.42 

cm. 
3.8±0.57 
9.8±0  63 

*  See  table  10  for  experimental  conditions. 

The  effect  of  this  treatment  upon  plant  growth  (table  11)  may  be 
summarized  briefly  as  follows :  When  the  oxygen  content  of  the  soil 
atmosphere  was  reduced  to  between  6  and  7  per  cent,  the  root  growth 
of  the  plants  was  only  about  one-third  to  one-half  that  of  the  control 
plants  which  had  a  soil  atmosphere  of  nearly  the  same  composition  as 
normal  air. 

Two  other  experiments,  both  with  culture  tubes,  were  conducted 
with  a  gas  mixture  of  low  oxygen  content.  In  the  first  experiment 
six-weeks-old  plants  were  used  and  the  tubes  sealed  with  a  paraffin- 
petrolatum  mixture  of  a  high  melting  point  (about  52°  C).  It  was 
found  necessary  to  reseal  the  treated  cultures  twice  during  the  experi- 
ment in  order  to  stop  leaks  around  the  plants.  This  was  done  with 
the  aid  of  a  hot  needle ;  unfortunately  some  of  the  plants  were  injured 
and  had  to  be  discarded.  Much  less  resealing  was  done  with  the  con- 
trol cultures.  The  gas  in  the  treated  tubes  was  changed  twice  daily 
by  drawing  through  each  tube  a  quantity  (85  cc. )  equal  to  about  three 
times  the  pore  space  occupied  by  the  soil  gases.  Growth  was  allowed 
to  take  place  for  15  days  at  a  temperature  of  25  ±  1°  C.  Analyses 
of  the  soil  gas  are  given  in  table  12. 


TABLE  12 
Composition  of  the  Soil  Gas  in  Tube  Cultures  with  Keduced  Oxygen  Supply 


Culture 

Day  of 

experimental 

period 

Number 

of 
analyses 

Per  cent 

co2 

Per  cent 
O2* 

Per  cent 

N2 

Gas-treated 

{  .! 

Average 

13 
13 
13 

0  9±0  05 

1  2±0  08 
1  6±0  09 

5  1±0  31 
3  8±0  22 
5  0±0  58 

94  0±0  28 

95  0±0.18 
93.4±0.54 

1  2±0  06 

4  6±0  21 

94  2±0  19 

Control 

Gas  from  tank 

15 

15 
5 

0  6±0  05 
0  2±0  02 

20.4±0.06 

3  0±0  03 

79.0±0  03 
96.8±0  05 

*  The  high  oxygen  content  of  the  treated  cultures,  as  compared  with  the  tank,  is  due  to  the  presence 
of  air  which  was  drawn  in  through  leaks  in  the  system  during  the  daily  gas  changes. 


108 


University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 


TABLE  13 
Growth  of  Sour-orange  Plants  in  Tube  Cultures  with  Low  Oxygen  Context* 

Temperature  25  ±  1°  C 


Num- 
ber of 
plants 

Stem 

Leaves 

Tap 
root 
elonga- 
tion 

Lateral  roots 

Total 

Culture 

Diam- 
eter 

Height 

Num- 
ber 

Fresh 
weight 

Num- 
ber 

Elonga- 
tion 

root 
elonga- 
tion 

Gas-treated     ,., 

20 
27 

cm. 
17±002 
16±  002 

cm. 
8  0±0  11 
8  2±0  10 

2.2±0  04 
2  6±0  13 

gm. 
12±  003 
13±  003 

cm. 
0  8±0  14 
3  3±0  25 

6  1±1  03 
8  6±0  55 

cm. 
4  6±0  89 
9  3±0.80 

cm. 
5  4±0.92 
12  6±0.91 

*  See  table  12  for  experimental  conditions 

The  effects  of  this  gas  mixture  upon  plant  growth  are  shown  in 
table  13.  The  data  indicate  that  the  total  root  elongation  of  the 
treated  plants  was  only  about  one-half  that  of  the  control  plants  when 
the  soil  atmosphere  consisted  of  4  to  5  per  cent  oxygen  and  the 
remainder  almost  exclusively  nitrogen. 

A  second  experiment  was  conducted  with  tube  cultures  using  a 
somewhat  higher  oxygen  content  (about  8%).  A  lower-melting  wax 
mixture  (m.p.  42°  C)  was  used  with  the  result  that  better  seals  were 
secured,  but  a  few  plants  were  injured,  apparently  by  paraffin  infiltra- 
tion. The  gas  in  the  treated  cultures  was  changed  twice  daily  by  draw- 
ing about  125  cubic  centimeters  through  each  tube.  A  temperature 
.of  25  ±  1°  C  was  maintained  during  the  experimental  period  of 
12  days. 

A  considerable  increase  of  carbon  dioxide  in  the  treated  cultures 
(table  14)  was  found  during  the  experiment.  It  is  thought  that  this 
increase  was  due  not  only  to  the  respiratory  activity  of  the  roots  but. 
also  to  some  residual  carbon  dioxide  in  the  water  in  the  gas  tank, 
which  gradually  escaped  from  solution  and  increased  the  carbon 
dioxide  content  of  the  c;as  mixture. 


TABLE  14 
Composition  of  the  Soil  Gas  in  Tube  Cultures   with    Diminished  Oxygen 

Supply 


Culture 

Day  of 

experimental 

period 

Number 
of 

analyses 

Per  cent 
C02* 

Per  cent 

Per  cent 
Ni 

{     I 

15 
15 
15 

1  2±0  08 

2  3±0  10 
4  8±0  14 

8  7±0  IS 
7  7±0  25 
7.7±0.38 

90.1±0  17 
90  0±0  21 

87.5±0  19 

2  8±0  19 

8  0±0  17 

89.2±0  18 

Control ... 

11 

15 

7 

0  6±0  05 
2.5±0  40 

20.3±0  04 
8  6±0  10 

79  1±0  01 
88  9±0  35 

*  See  text  for  explanation  of  the  increasing  carbon  dioxide  percentages. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         109 

TABLE  15 
Growth  of  Sour-orange  Plants  in  Tube  Cultures  with  Low  Oxygen  Content* 

Temperature    25  ±  1°  C 


Num- 
ber of 

plants 

Stem 

Leaves 

Tap 
root 
elonga- 
tion 

Lateral  roots 

Total 

Culture 

Diam- 
eter 

Height 

Num- 
ber 

Fresh 
weight 

Num- 
ber 

Elonga- 
tion 

root 
elonga- 
tion 

Gas-treated 

26 
26 

cm. 
.17±  002 
16±002 

cm. 
7  5±0.11 
7.6±0  09 

2.1±0  01 
2.3±0.05 

gm. 
ll±.0O2 
11±003 

cm. 
0.8±0  14 
1  8±0.24 

5  0±0  63 
7.5±0  59 

cm. 

2  1±0.32 
4  5±0  44 

cm. 
2  9±0  44 
6  3±0  61 

*  See  table  14  for  experimental  conditions. 

A  considerable  retardation  of  root  growth  was  again  evident,  even 
with  an  average  oxygen  content  of  8  per  cent  (table  15).  It  was  sur- 
prising to  find  that  the  hydrogen-ion  concentration  of  the  extracted 
solution  was  very  low,  pH  8.5  for  the  treated  cultures  and  pH  9.2  for 
the  control  cultures.  It  was  pointed  out  to  the  writer  that  the  nature 
of  the  minerals  found  in  the  soils  of  the  region  from  which  the  sand 
was  obtained  is  such  as  to  give  the  soil  solution  an  alkaline  reaction. 
This  may  account  for  the  high  pH  values  obtained  for  the  solutions 
withdrawn  from  the  sand  cultures.  Apparently  the  greater  carbon 
dioxide  content  of  the  treated  cultures  was  responsible  for  their  more 
acid  reaction. 


GKOWTH  IN  HIGH  CONCENTEATIONS  OF  CAEBON  DIOXIDE 
It  was  desired  to  determine  whether  carbon  dioxide  exerts  a  retard- 
ing influence  upon  the  growth  of  Citrus  plants.  In  order  that  oxygen 
might  not  be  a  limiting  factor,  it  appeared  desirable  to  supply  the  same 
quantity  of  oxygen  to  the  treated  cultures  as  the  controls  would 
receive  from  the  surrounding  air.  Six-weeks-old  sour-orange  seedlings 
were  grown  in  culture  tubes  with  a  soil  atmosphere  of  high  carbon 
dioxide  (55%)  and  normal  oxygen  content  for  15  days.  The  tem- 
perature during  this  period  was  maintained  at  25  ±  1°  C.  A  wax 
with  a  rather  high  melting  point  was  used  for  sealing  in  the  plants 
and  it  again  proved  necessary  to  resort  to  the  resealing  of  leaky 
cultures.  This  resulted  in  injury  to  some  of  the  plants,  particularly 
with  the  treated  cultures  which  were  resealed  more  often,  so  that  many 
of  the  plants  had  to  be  discarded. 

A  considerably  lower  carbon  dioxide  content  was  found  in  the 
treated  cultures  than  existed  in  the  tank  (table  16).  This  and  the 
high  probable  errors  of  the  tank  analyses,  were  evidently  the  result 


110 


University  of  California  Publications  in  Agricultural  Sciences      [Vol.5 


of  dilution  by  the  outside  air  which  was  drawn  into  the  system  through 
occasional  leaks.  The  rather  large  probable  errors  in  the  tank  analyses 
were  due  to  a  fluctuating  composition  associated  with  occasional 
additions  of  quantities  of  gas. 


TABLE  16 
Composition  of  Soil  Gas  in  the  Tube  Cultures  with  Increased  Carbon 

Dioxide  Supply 


Culture 

Day  of 

experimental 
period 

Number 

of 
analyses 

Per  cent 

co2* 

Per  cent 
02 

Per  cent 
N? 

Gas-treated 

!   i 

13 
13 
13 

53.3±2  03 

54  6dz2  80 

55  7±2.37 

20.4±0  16 
19.9±0.18 
18.3dz0.38 

26.3±2  08 
25.5±2  89 

26  0±2  11 

54.5dbl.34 

19.5dz0  17 

26  Odzl  32 

Control 

13 

14 
12 

0  4±0  03 
74.2dz0.41 

20.5dzO  03 
20.7dz0.43 

79.ldz0.01 
5  ldzO  35 

*  See  text  for  explanation  of  the  low  carbon  dioxide  content  of  the  treated  cultures  as  compared  to 
the  tank. 

TABLE  17 
Growth  of  Sour-orange  Plants  in  Tube  Cultures  with  High  Carbon  Dioxide 

Content* 
Temperature  25  ±  1°  C 


Num- 
ber of 
plants 

Stem 

Leaves 

Tap 
root 
elonga- 
tion 

Lateral  roots 

Total 

Culture 

Diam- 
eter 

Height 

Num- 
ber 

Fresh 
weight 

Num- 
ber 

Elonga- 
tion 

root 
elonga- 
tion 

Gas-treated 
Control 

19 
25 

cm. 
13dz  002 
15dz  002 

cm. 
9.3dzO  16 
9.7zb0.12 

2  OdzO  00 
2  8dz0  12 

gm. 

09±  004 
12±  005 

cm. 
0  08±0  02 
3  4±0  35 

0  IzbO  04 
9.3dzO  46 

cm. 

0  01 dz  004 

7.7dzO  63 

cm. 
0  09dz0  02 
ll.ldz0.75 

*  See  table  16  for  experimental  conditions. 

The  data  presented  in  table  17  indicate  that  growth  was  prac- 
tically suppressed  by  the  large  percentage  of  carbon  dioxide  in  the 
soil  atmosphere  of  the  treated  cultures.  It  should  be  remembered, 
however,  that  the  effects  produced  by  the  necessary  resealing  may  be 
a  contributing  factor  in  the  case  of  the  treated  plants  retained. 

In  a  second  experiment  of  this  type  a  wax  with  a  lower  melting 
point  (m.p.  about  35°  C)  was  used  for  sealing  around  the  plants. 
Although  the  resulting  seals  were  fairly  effective,  the  mixture  showed 
evidence  of  infiltrating  into  some  of  the  plant  stems  so  that  the  experi- 
ment had  to  be  terminated  on  the  eighth  day.  The  experimental  con- 
ditions were  in  general  the  same  as  those  for  the  previous  experiment . 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         111 

including  semi-daily  gas  renewals  for  the  treated  cultures,  except  that 
a  lower  carbon  dioxide  content  was  employed.  The  data  for  this 
experiment  are  briefly  summarized  in  table  18. 

TABLE  18 
Growth   or    Sour-orange   Plants   in    Tube   Cultures    with    a    High    Carbon 

Dioxide  Content 
Temperature  25  ±  1°  C 


Gas  content 

Num- 
ber of 
plants 

Tap 
root 
elonga- 
tion 

Lateral  roots 

Total 

Culture 

Per  cent 
C02 

Per  cent 

o2 

Per  cent 

N2 

Number " 

Elonga- 
tion 

root 
elonga- 
tion 

Gas-treated 
Control 

37  4±1  06 
0.4±0.02 

16  5±0  15 
20.5±0  02 

46  1±1  04 
79.1±0  02 

20 
27 

cm. 

0  0 

0  6±0  11 

0  0 
2  6±0  59 

cm. 

0  0 

0.6±0.13 

cm. 

0  0 

1.2±0.23 

In  general  it  may  be  concluded  that  the  presence  of  high  concen- 
trations of  carbon  dioxide  in  the  soil  atmosphere  resulted  in  a  definite 
retarding  effect  upon  the  root  elongation  of  sour-orange  seedlings. 
The  fact  that  some  of  the  treated  plants  died  during  the  course  of 
the  experiment,  while  those  discarded  from  the  controls  were  only 
injured,  suggests  that  the  carbon  dioxide  was  an  important  factor  in 
the  death  of  some  of  the  treated  plants. 


DISCUSSION 
The  results  of  the  soil-gas  studies  presented  in  this  section  may  be 
briefly  summarized  in  the  following  statements :  Check  experiments 
conducted  with  gaseous  mixtures  similar  to  that  of  the  air  showed  that 
normal  growth  (as  compared  with  control  plants)  may  be  secured 
under  the  conditions  of  the  experiment.  Treatments,  in  which  soil 
gases  of  diminished  oxygen  content  were  employed,  resulted  in  a  con- 
siderable reduction  of  root  growth.  For  example,  the  root  elongation 
in  gaseous  mixtures  containing  5  or  8  per  cent  oxygen  and  the 
remainder  chiefly  nitrogen  was  only  about  one-half  that  of  the  control 
plants  at  a  temperature  of  25°  C.  A  further  reduction  of  the  oxygen 
content  to  a  value  of  1.2  to  1.5  per  cent  resulted  in  the  complete  sup- 
pression of  root  growth  at  28°  C.  Finally,  the  presence  of  a  high 
carbon  dioxide  content  (37  to  55%)  in  the  soil  gas  was  found  to  result 
in  an  almost  complete  suppression  of  root  growth  at  a  temperature  of 
25°  C,  even  though  the  oxygen  content  approached  that  of  normal  air. 
Not  only  was  root  growth  inhibited  but  some  plants  showed  definite 
injury  which  was  apparently  due  to  the  carbon  dioxide  treatment. 


112  University  of  California  Publications  in  Agricultural  Sciences      [Vol.  5 

The  question  arose  whether  the  treatment  with  carbon  dioxide 
gave  the  solution  an  acid  reaction  which  in  turn  resulted  in  sup- 
pressing root  growth.  In  order  to  test  this  possibility  ten  culture  tubes 
were  set  up  as  described  for  the  soil-gas  studies,  but  without  plants. 
After  semi-daily  gas  treatments  for  a  period  of  four  days  with  a  gas 
mixture  containing  a  high  carbon  dioxide  concentration,  the  gas  con- 
tents of  the  tubes  were  analyzed  and  the  pH  values  of  the  extracted 
solutions  determined.  The  average  gas  composition  for  the  ten  tubes 
was  70.2  per  cent  carbon  dioxide,  5.5  per  cent  oxygen,  and  24.3  per  cent 
nitrogen.  The  pH  determinations  showed  an  average  value  of 
7.56  ±  0.02.  It  is  therefore  evident  that  the  reaction  of  the  soil  solu- 
tion was  not  the  factor  limiting  root  growth,  since  the  hydrogen-ion 
concentration  of  the  gas-treated  cultures  was  nearer  the  optimum  for 
growth  than  that  of  cultures  having  only  a  small  amount  of  carbon 
dioxide  and  previously  found  to  be  decidedly  alkaline  (8.5  to  9.2). 

The  results  obtained  in  these  studies  agree,  in  general,  with  those 
obtained  by  different  investigators  for  various  plants.  Cannon  (1925) 
has  worked  extensively  in  this  field  and  has  studied  the  soil-gas  rela- 
tions of  a  large  number  of  plants.  His  findings  indicate  that,  while 
considerable  difference  of  behavior  existed  with  various  species,  prac- 
tically all  required  an  appreciable  amount  of  oxygen  present  in  the 
soil  for  continued  root  growth.  Furthermore,  it  was  found  that  the 
oxygen  requirement  for  root  growth  increased  with  temperature,  i.e., 
an  oxygen  content  which  permitted  normal  root  growth  at  a  given 
temperature  might  act  to  limit  growth  at  a  higher  temperature.  In 
the  same  way  it  was  observed  that  different  plants  reacted  somewhat 
differently  toward  large  concentrations  of  carbon  dioxide  in  the  soil 
atmosphere,  and  that  such  concentrations  might  prove  toxic  for  many 
plants.  For  example,  the  growth  of  Opuntia  was  entirely  checked  by 
exposure  to  a  mixture  of  25  per  cent  oxygen  and  75  per  cent  carbon 
dioxide  for  a  short  period,  but  growth  was  quickly  resumed  with  the 
admission  of  atmospheric  air.  Varying  concentrations  of  carbon 
dioxide  diluted  with  air  or  oxygen  were  found  to  depress  root  growth 
in  Covillea,  Prosopis,  Opuntia,  and  Krameria. 

In  addition,  Cannon  has  recorded  the  results  of  some  experiments 
with  Citrus  plants.  He  reports  that  sweet-orange  plants  were  able 
to  withstand  high  concentrations  of  carbon  dioxide  (75%  C02  and 
25%  02  at  20  to  25°  C)  for  a  period  of  four  days,  but  that  the  result- 
ing growth  was  slow.  Cannon  states  that,  in  the  absence  of  carbon 
dioxide,  sweet-orange  and  rough-lemon  plants  gave  evidence  of  con- 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         113 

tinued  root  growth  with  2.5  per  cent  oxygen,  but  that  root  growth  was 
inhibited  when  the  oxygen  content  was  reduced  to  2.0  per  cent.  The 
minimum  oxygen  content  for  root  growth  with  sour-orange  plants  was 
thought  to  be  somewhat  lower  than  that  for  the  sweet  orange.  These 
values  wTere  obtained  for  short  experimental  periods  (1  to  5  days)  and 
represent  measurements  made  with  a  comparatively  small  number  of 
plants  giving  results  of  a  doubtful  statistical  reliability. 

Noyes,  Trost,  and  Yoder  (1918)  have  also  studied  the  effects  of 
high  concentrations  of  carbon  dioxide  upon  the  growth  of  various 
plants.  The  continuous  passage  of  carbon  dioxide  through  the  soil  in 
which  Christmas  pepper  (Capsicum  annum  abbreviatum)  plants  were 
growing  resulted  in  a  stunting  and  dwarfing  of  the  main  roots.  An 
intermittent  treatment  produced  coarser  and  more  'clumped'  roots 
than  those  of  the  control.  Other  plants  studied  showed  somewhat 
similar  effects  but  to  a  slighter  degree. 

The  effects  of  these  treatments  upon  the  growth  of  plant  roots 
must  evidently  depend  upon  a  fundamental  process  (or  processes) 
going  on  within  the  plant  tissues.  It  has  been  suggested  by  Livingston 
and  Free  (1917)  that  the  process  of  respiration  is  the  one  primarily 
concerned  in  this  connection.  The  effect  of  a  low  content  of  available 
oxygen  would  therefore  be  to  retard  aerobic  respiration  by  causing 
oxygen  to  become  a  limiting  factor. 


GENERAL  DISCUSSION 

It  seems  evident  from  the  experimental  results  obtained  that  the 
root  growth  of  Citrus  seedlings  may  be  markedly  influenced  by 
changes  in  certain  of  the  environmental  factors.  Extreme  changes  in 
any  one  factor  may  result  in  profound  changes  in  plant  growth. 

Moderate  changes  in  one  or  more  of  the  environmental  factors, 
however,  may  or  may  not  produce  significant  changes  in  plant  growth. 
When  these  variations  occur  well  within  the  range  favorable  to  plant 
growth,  the  response  of  the  plant  is  likely  to  be  small.  When,  on  the 
other  hand,  the  changes  are  near  either  extreme  of  the  range,  the 
response  appears  to  be  greater.  Thus  temperatures  of  27°  and  31°  C 
were  associated  with  values  of  20.5  and  19.9  centimeters  respectively, 
for  the  average  root  elongation  obtained  with  populations  of  30  sour- 
orange  plants.  But  an  increase  from  13°  to  18°  C  was  accompanied 
by  an  increased  root  elongation  of  13.5  centimeters,  and  an  increase 
from  34°  to  37°  C  resulted  in  a  reduction  of  root  elongation  from 
10.2  to  1.4  centimeters. 


114  University  of  Calif ornia  Publications  in  Agricultural  Sciences      [Vol.  .3 

It  is  of  interest  to  apply  the  findings  of  this  investigation  to  an 
interpretation  of  growth  under  field  conditions.  For  example,  unpub- 
lished data  collected  by  the  Division  of  Orchard  Management  of  the 
Citrus  Experiment  Station  give  46°  F  (approximately  8°  C)  as  the 
minimum  soil  temperature  and  89°  F  (approximately  32°  C)  as  the 
maximum  for  1925.  These  temperatures  were  recorded  for  a  soil 
depth  of  one  foot  at  Riverside,  California.  It  is  evident  that  they 
represent  a  range  including  both  subminimal  and  superoptimal  tem- 
peratures for  root  elongation. 

A  second  important  factor  is  the  reaction  of  the  soil  solution. 
Usually  the  reaction  of  the  soils  in  the  citrus  regions  of  southern 
California  is  definitely  alkaline.  In  certain  cases  soil  alkalinity  may 
be  the  factor  limiting  growth,  and  perhaps,  to  a  still  greater  degree, 
limiting  root-hair  production. 

In  view  of  the  responses  to  increased  aeration  obtained  with  sour- 
orange  roots,  it  seems  evident  that  the  aeration  of  the  soil  must  be  an 
important  factor  influencing  root  growth  under  field  conditions.  Thus, 
such  conditions  as  a  'tight'  soil,  or  the  occurrence  of  a  plow-sole,  irri- 
gation hardpan,  or  other  compacted  soil  layers  which  act  to  restrict  soil 
aeration,  may  undoubtedly  exert  a  profound  influence  upon  root 
growth.  The  application  of  excessive  amounts  of  water  to  the  soil 
also  acts  to  restrict  aeration.  The  respiratory  activity  of  the  roots 
under  such  conditions  results  in  a  soil  air  poor  in  oxygen  and  rich  in 
carbon  dioxide.  Both  of  these  conditions  may  operate  to  limit  the 
growth  of  sour-orange  roots.  On  the  other  hand,  the  situation  occur- 
ring in  well  aerated,  well  drained  soils  undoubtedly  leads  to  maximum 
root  growth,  other  conditions  being  favorable. 

In  general  it  is  evident  that  root  growth  in  the  field  is  conditioned 
by  various  factors  which  may  be  closely  interrelated  and  may  often 
operate  in  a  very  complex  manner.  The  experimental  results  herein 
described,  and  their  application  to  field  conditions,  must  therefore  be 
qualified  by  making  allowance  for  the  special  experimental  conditions 
and  for  the  more  complex  situation  associated  with  the  growth  of 
plants  in  the  field. 

SUMMARY 

1.  The  root  growth  of  seedlings  of  grapefruit,  sour-orange,  and 
sweet-orange  in  solution  cultures  was  found  to  have  a  minimum  tem- 
perature of  approximately  12°  C,  an  optimum  temperature  of  26°  C, 
and  a  maximum  temperature  of  approximately  37°  C. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         115 

2.  The  optimum  temperature  for  the  production  of  root  hairs  for 
sweet-  and  sour-orange  plants  was  apparently  somewhat  higher  than 
that  most  favorable  to  root  elongation — approximately  33°  C  as 
contrasted  with  26°  C. 

3.  The  root  growth  of  sour-orange  seedlings  in  solution  cultures 
gave  evidence  of  being  significantly  influenced  by  the  reaction  of  the 
solution.  When  the  hydrogen-ion  concentrations  were  adjusted  twice 
daily  it  was  found  that  a  minimum  of  root  elongation  occurred  at  or 
below  pH  4.0,  a  maximum  at  pH  6.5  (or  approximate  neutrality),  and 
a  second  minimum  at  or  above  pH  9.0. 

4.  The  hydrogen-ion  concentration  most  favorable  for  the  produc- 
tion of  root  hairs  was  found  to  be  distinctly  acid,  pH  5.0. 

5.  A  continuous  aeration  of  the  culture  solution  was  found  to  give 
increased  root  growth  as  measured  by  the  elongation  of  the  tap  and 
lateral  roots. 

6.  The  production  of  root  hairs  was  greatly  enhanced  by  aeration. 
The  data  indicate  that  root-hair  formation  was  much  more  responsive 
to  the  aeration  treatment  than  was  root  elongation. 

7.  Data  secured  from  sour-orange  seedlings  grown  in  sand  cultures 
indicate  that  root  elongation  was  entirely  suppressed  by  a  very  low 
oxygen  content  of  the  soil  atmosphere  (1.2  to  1.5%  at  28°  C),  and 
that  a  retarding  influence  was  evident  when  the  oxygen  content  was 
considerably  higher  (5  to  8%). 

8.  The  root  growth  of  sour-orange  plants  in  sand  cultures  was  also 
found  to  be  adversely  affected  by  high  concentrations  of  carbon 
dioxide  in  the  soil  atmosphere.  Total  root  elongation  was  suppressed 
with  37  to  55  per  cent  carbon  dioxide  at  a  temperature  of  25°  C,  even 
though  the  oxygen  content  (17  to  20%)  of  the  soil  atmosphere  was 
not  greatly  below  that  of  normal  air. 

The  writer's  thanks  are  extended  to  Dr.  H.  J.  Webber  with  whom 
the  work  was  initiated  and  to  Dr.  H.  S.  Reed  under  whose  helpful 
direction  the  work  was  completed.  Thanks  are  also  due  to  Dr.  A.  R.  C. 
Haas  for  valuable  aid  in  the  final  preparation  of  this  paper. 


116  University  of  California  Publications  in  Agricultural  Sciences      [Vol.5 


LITERATURE  CITED 

Allison,  E.  V. 

1922.  The  relation  of  aeration  to  the  development  of  the  soy  bean  plant  in 

artificial   culture.      N.   J.   Agr.   Exp.   Sta.   Ann.   Kept.    (1921),   pp. 
338-45. 

Allison,  E.  V.,  and  Shive,  J.  W. 

1923.  Studies  on  the  relation  of  aeration  and   continuous  renewal  of  the 

nutrient   solutions  to  the  growth  of  soy  beans  in  artificial  culture. 
Am.  Jour.  Bot.,  vol.  10,  pp.  554-66. 
1923a.  Micro-sampling    for    the    determination    of    dissolved    oxygen.      Soil 
Sei.,  vol.  15,  pp.  489-91. 

Andrews,  F.  M. 

1920.  The  effect  of  aeration  on  plants.  Proc.  Ind.  Acad.  Sci.  (1920),  pp. 
147-48. 

Andrews,  F.  M.,  and  Beals,  C.  0. 

1919.  The  effect  of  soaking  in  water  and  of  aeration  on  the  growth  of  Zea 

mais.    Bull.  Torr.  Bot.  Club,  vol.  44,  pp.  91-100. 

Bergman,  H.  F. 

1920.  The  relation  of  aeration  to  the  growth  and  activity  of  roots  and  its 

influence   on  the   ecesis   of  plants   in   swamps.     Ann.   Bot.,   vol.   34, 
pp.  13-33. 

Cannon,  W.  A. 

1925.  Physiological  features  of  roots,  with  especial  reference  to  the  rela- 
tion of  roots  to  aeration  of.  the  soil.  Carnegie  Inst.  Publ.  368, 
pp.  1-168. 

Fawcett,  H.  S. 

1921.  The    temperature    relations    of    growth    in    certain    parasitic    fungi. 

Univ.  Calif.  Publ.  Agr.  Sci.,  vol.  4,  pp.  183-232. 

Free,  E.  E. 

1917.  The  effects  of  aeration  upon  the  growth  of  buckwheat  in  solution 
cultures.     Johns  Hopkins  Univ.  Circ.  n.  s.,  vol.  3,  pp.  198-99. 

Haldane,  J.  S. 

1920.     Methods  of  air   analysis.      London. 

Knight,  E.  C. 

1924.  The  response  of  plants  in   soil-  and  in  water-culture  to  aeration  of 

the  roots.  Ann.  Bot.,  vol.  38,  pp.  305-25. 

Landolt,  H.,  BOrnstein,  E.,  and  Eoth,  W. 

1912.     Landolt-Bornstein  physikalischen-chemische  Tabellcn.     Berlin. 

Livingston,  B.  E.,  and  Fawcett,  H.  S. 

1920.  A  battery  of  chambers  with  different  automatically  maintained  tem- 
peratures.    Phytopath,  vol.  10,  pp.  336-40. 


1927]     Girton:  Growth  of  Citrus  Seedlings  as  Influenced  by  Environment         117 

Noyes,  H.  A.,  Trost,  J.  F.,  and  Yoder,  L. 

1918.  Eoot  variations  induced  by  carbon  dioxide  gas  additions  to  soil. 
Bot.  Gaz.,  vol.  66,  pp.  364-73. 

Peltier,  G.  L. 

1920.  Influence  of  temperature  and  humidity  on  the  growth  of  Pseudomonas 
citri  and  its  host  plants  and  on  infection  and  development  of  the 
disease.     Jour.  Agr.  Res.,  vol.  20,  pp.  447-506. 

Schwarz,  Franz 

1883.  Die  Wurzelhaare  der  Pflanzen.  Untersuch.  Bot.  Inst.  Tubingen, 
vol.  1,  pp.  135-88. 

Snow,  Laetitia  M. 

1905.     The  development  of  root  hairs.    Bot.  Gaz.,  vol.  40,  pp.  12-48. 

Stiles,  W.,  and  Jorgensen,  I. 

1917.  Observations  on  the  influence  of  aeration  of  the  nutrient  solution  in 
water  culture  experiments.    New  Phytol.,  vol.  16,  pp.  181-97. 

Treadwell,  F.  P.,  and  Hall,  W.  T. 

1915.     Analytical  Chemistry,  vol.  2.     New  York. 


h 


